Bacterial transposons such as Tn5 evolved within the cell by maintaining a low mobility level. While necessary for the transposon to survive, the low mobility level has inhibited the ability of researchers to detail the molecular transposition process and to exploit the transposition process for use, e.g., in the development of new diagnostic and therapeutic resources. Tn5 is a conservative “cut and paste” transposon of the IS4 family (Rezsohazy, R., Hallet, B., Delcour, J., and Mahillon, J., “The IS4 family of insertion sequences: evidence for a conserved transposase motif,” Mol Microbiol. 9:1283–1295 (1993)) that encodes a 53 kD transposase protein (Tnp) that is responsible for its movement. The wild-type Tn5 transposase (Tnp) amino acid and nucleic acid sequences are known (Ahmed, A. and Podemski, L. The Revised Sequence of Tn5. Gene 154(1),129–130(1995), incorporated by reference as if set forth herein in its entirety). A nucleic acid sequence that encodes wild-type Tn5 Tnp is attached as SEQ ID NO:1. A polypeptide sequence encoded by SEQ ID NO:1 which corresponds to wild-type Tn5 Tnp is attached as SEQ ID NO:2.
The Tnp protein facilitates movement of the entire element by binding initially to each of two 19 bp specific binding sequences termed outside end (OE; SEQ ID NO:3), followed by formation of a nucleoprotein structure termed a synapse, blunt ended cleavage of each end, association with a target DNA, and then strand transfer (Reznikoff, W. S., Bhasin, A., Davies, D. R., Goryshin, I. Y., Mahnke, L. A., Naumann, T., Rayment, I., Steiniger-White, M., and Twining, S. S., “Tn5: A molecular window on transposition,” Biochem. Biophys. Res. Commun. 266:729–34 (1999)). Tn5 Tnp can also promote movement of a single insertion sequence by using a combination of OE and inside end (IE; SEQ ID NO:4) sequences. The IE is also 19 bp long and is identical to OE at 12 of 19 positions. In vivo, Tn5 Tnp exhibits a marked preference for OE in E. coli. Transposase recognition and binding to IE is inhibited in E. coli by the presence of four dam methylation sites (GATC palindromes) which add four methyl groups per inside end sequence (IEME; also depicted as SEQ ID NO:4, methylation not shown) (Yin, J. C. P., Krebs, M. P., and Reznikoff, W. S., “Effect of dam Methylation on Tn5 Transposition,” J. Mol Biol., 199:35–45 (1988), incorporated by reference as if set forth herein in its entirety). This methylation reduces tranhsposition by reducing protein-DNA primary recognition (Jilk, R. A., York, D., and Reznikoff, W. S., “The organization of the outside end of transposon Tn5, ” J. Bacteriol. 178:1671–1679 (1996)).
Tn5 transposon also encodes an inhibitor protein that can interfere with transposase activity. The inhibitor-encoding sequence overlaps with the sequence that encodes the transposase. An AUG in the wild-type Tn5 Tnp gene that encodes methionine at transposase amino acid 56 is the first codon of the inhibitor protein. Replacement of the methionine at position 56 with an alanine has no apparent effect upon the transposase activity. However, it prevents translation of the inhibitor protein and thus results in a higher transposition rate. Weigand, T. W. and W. S. Reznikoff, “Characterization of Two Hypertransposing Tn5 Mutants,” J. Bact. 174:1229–1239 (1992), incorporated herein by reference.
A principal roadblock to understanding how Tn5 Tnp works is the fact that purified wild-type Tnp has no detectable activity in vitro. Recently, a double mutant hyperactive form of transposase (“Tnp EK/LP”) that promotes the transposition reaction in vitro was developed (U.S. Pat. No. 5,965,443, incorporated herein by reference in its entirety). The Tnp EK/LP protein differs from wild-type Tn5 Tnp at position 54 (Glu to Lys mutation) and at position 372 (Leu to Pro mutation), in addition to a non-essential but advantageous change at position 56 that prevents production of the inhibitor protein. The modified hyperactive Tnp protein increases the dramatic preference for OE termini of wild-type Tn5 Tnp. In addition, certain modifications on the OE sequence have been shown to increase the transposition frequency by Tnp EK/LP (U.S. Pat. No. 5,925,545 and U.S. Pat. No. 6,437,109, both of which are herein incorporated by reference in their entirety). Tnp EK/LP has clarified many aspects of TnS transposition that were not previously adequately addressable in vivo.
Another recent development in Tn5 research involves the identification of Tn5 mutants that have a higher avidity for IE than OE sequences (U.S. Pat. No. 6,406,896, which is herein incorporated by reference in its entirety). These mutants contain a mutation at amino acid position 58 and can further contain a mutation at amino acid position 8, 344, or both. Both unmethylated and methylated IE (IEME) sequences can be used efficiently for transposition by these Tn5 mutants.
In vitro polynucleotide transposition is a powerful tool for introducing random or targeted mutations into a genome. Useful in vitro transposition systems based upon the Tn5 transposon are disclosed in U.S. Pat. No.5,948,622, 6,159,736 and U.S. Pat. No. 6,406,896, all of which are incorporated herein by reference in their entirety.